Allelic losses on chromosome arm 3p are the most frequent and earliest genetic abnormality detected in human lung cancers, indicating the presence of one or more lung cancer tumor suppressor genes (TSGs) in this region. In particular, a 600-kb lung cancer homozygous deletion region at 3p21.3 shows frequent allele loss in tumors and smoking-damaged lung epithelium. We identified a group of 23 candidate TSGs in this small 600-kb 3p21.3 region, several of which occasionally suffer mutations, but no one gene was targeted. Several (CACNA2D2, BLU, RASSFIA isoform, and SEMA3B) had their expression extinguished related to tumor-acquired promoter DNA methylation. Using a variety of expression vectors, we found that several of the candidate 3p21.3 TSGs, FUS1, 101F6, NPRL2, and SEMA3B, effectively inhibited tumor cell growth by induction of apoptosis in vitro and in vivo, while others, RASSF1A and CACNA2D2, efficiently inhibited anchorage-independent growth in vitro and xenograft growth in vivo without inducing apoptosis (RASSF1A) or inducing apoptosis only in p53 wild-type tumors (CACNA2D2), strongly suggesting that several of these contiguous 3p21.3 genes function as lung cancer tumor suppressors. The purpose of this project is to gain a comprehensive understanding of the molecular mechanisms and biological pathways of the products of the 3p21.3 genes alone and in combination with each other and other TSGs with the goal of developing these genes into new therapeutics and diagnostics. The Specific Aims are 1. to confirm the loss of expression of these genes and the timing of this loss in lung cancer pathogenesis by immunohistochemical analysis of tumor and preneoplastic tissues using tissue microarrays (TMAs) and new proteomics tools (ProteinChip, Ciphergen Biosystems, Fremont, CA) to study modifications of their proteins in these samples; 2a. to determine the molecular changes and cellular responses (particularly induction of apoptosis) in human NSCLC cells mediated by re-expression of these 3p2!.3 genes, p53, and FHIT using mRNA (microarray) expression profiling and SELDI-Mass spectrometry analysis-based protein profiling; 2b. to confirm the role of these 3p21.3-encoded proteins as tumor suppressors by knocking down their expression using siRNA technology in normal human epithelial cultures and then studying the resultant mRNA and protein expression profiles and testing for the development of the malignant phenotype; 3. to quantitatively evaluate interactions of the 3p21.3 genes in combination with each other and with p53 or FHIT for their tumor-suppressing activities in vitro and in vivo; 4. to conduct a sehes of Phase I clinical tdals to evaluate these genes (starting with FUS1) as therapeutic agents delivered systemically using a DOTAP:Chol lipoplex. By studying the same materials as SPORE Projects 2 and 3, we will be able to integrate the data from the different projects. Thus, this project has a "pipeline" of candidate TSGs that we propose to bring through preclinical tests into clinical therapeutic trials singly or in combination as new therapies for lung cancer; the project will interface with other SPORE Projects to exploit information obtained from the trials for diagnosis, prognosis and risk assessment purposes.